The process of autophagy, or bulk degradation of cellular proteins through an evolutionarily conserved autophagosomic-lysosomal pathway, is important in survival during nutrient stress, differentiation and development, and negative growth control. However, almost nothing is known about the role of this cellular pathway in defense against intracellular pathogens. The broad objective of this proposal is to evaluate the novel hypothesis that autophagy functions as an intracellular antiviral pathway that degrades viral particles and is antagonized by viral gene products required for disease pathogenesis. In support of this hypothesis, we have previously shown that the first identified mammalian autophagy gene, beclin 1, inhibits Sindbis virus replication and protects mice against lethal Sindbis virus encephalitis. In addition, our preliminary data demonstrate that the well-characterized IFN-inducible antiviral molecule, PKR, is required for herpes simplex virus (HSV)-induced autophagy and that the HSV-1-encoded neurovirulence gene product, ICP34.5, antagonizes PKR-dependent autophagy. In this proposal, there are three specific aims that investigate three major related hypotheses, including: (1) Autophagy is a novel IFN-regulated host pathway that is activated in HSV-1 infection and requires eIF2a kinase signaling and the beclin 1 autophagy gene; (2) Autophagy functions as an antiviral host defense pathway by facilitating the degradation of intracytoplasmic viral particles, viral proteins, and/or viral RNA; and (3) the ICP34.5 gene product regulates HSV-1 neurovirulence by antagonizing the autophagy function of PKR and/or Beclin 1. In the first specific aim, we will measure autophagy in wild-type HSV-1 and HSV-1 delta34.5-infected MEFs and neurons derived from pkr, beclin 1, and IFNa/bR knockout mice. In the second specific aim, we will examine the effects of PKR and Beclin 1-dependent autophagy on the replication and life cycle of HSV-1 and rates of viral protein and viral RNA degradation. In the third specific aim, we will use a yeast system to perform structure-function analyses of the effects of ICP34.5 on PKR and Beclin 1-dependent autophagy. This information will be used to generate new HSV- 1 ICP34.5 mutant viruses for analyses of the interrelationships between in vitro antagonism of translational block, in vitro antagonism of autophagy, and in vivo neurovirulence. These studies will provide novel insights about the role of autophagy in antiviral host defense and the role of viral evasion of autophagy in neurovirulence.